Project Summary/Abstract The central goal of this project is to develop strategies to epigenetically reprogram pancreatic cancer cells to diminish metastatic spread. This objective is based on our recent demonstration that pancreatic cancer metastasis is accompanied by a stereotypical pattern of enhancer activation. We implicated the pioneer transcription factor FOXA1 as a driver of enhancer reprogramming and of metastatic spread in this context. In this proposal, we seek to define the enhancer-metastasis connection. In doing so, we seek to nominate a new class of epigenetic targets, which might be uniquely capable of eliminating metastatic potential. In the first Aim of this proposal, we will employ a functional genomics approach to perturb every FOXA1-regulated gene and enhancer and determine whether FOXA1-dependent metastasis can be suppressed. This approach will take advantage of our recent innovations in domain-focused CRISPR screening and will deepen our understanding of the pro-metastatic components of this epigenetic program. In the second Aim of this proposal, we will investigate the earliest steps of the enhancer reprogramming process that occur prior to FOXA1 upregulation. This effort builds from our unexpected observation that metastasis-specific enhancers are already present in an accessible chromatin state in pre-metastatic pancreatic tumor cells. This suggests that additional molecular events occur prior to FOXA1 upregulation to set the stage for enhancer reprogramming during metastasis. We will investigate the transcription factor FOXA2, which our experiments suggest is the critical bookmark that opens up metastasis-specific enhancers in pre-metastatic cancer cells. In addition, we will determine how repressive Polycomb complexes act to restrain enhancer activation prior to FOXA1 upregulation. By evaluating the consequences of FOXA2/Polycomb perturbation, we will provide a proof-of-concept that metastasis- associated enhancers can be effaced at early stages of pancreatic cancer progression. The final Aim of this proposal will be to extend our enhancer mapping studies into the squamous-subtype of pancreatic cancer, which is a recently defined disease entity associated with a particularly dismal prognosis. We will employ a newly characterized complement of patient-derived pancreatic cancer organoids to compare enhancer profiles of squamous-subtype versus the more classical form of this disease. We will identify master-regulators of this squamous transcriptional program, and perform genetic experiments to determine the role of such factors in promoting tumor progression and metastatic spread. We will also determine whether squamous cell identity in PDA is associated with unique epigenetic dependencies. Collectively, the proposed research will provide a mechanistic framework for developing epigenetic therapies that target the unique enhancer configuration of metastatic pancreatic cancer cells.